Team:REC-CHENNAI/Project-Motivation
PROJECT MOTIVATION
Although a broad-spectrum transfection reagent, Lipofectamine 2000 displayed a positive correlation between cytotoxicity and transfection efficiency. [1] The transfection efficiency of Lipofectamine 2000 was compromised by its high toxicity, which may adversely affect its application in most cells. The cytotoxicity exhibited by Lipofectamine 2000 could result in the non-specific activation/inactivation of certain genes and affect the functional assays scheduled post-transfection. A recent study has demonstrated that the delivery of negative control siRNA using Lipofectamine 2000, a commonly used cationic lipid transfection reagent, increased the number of autophagosomes in Huh7.5 cells in a dose- and time-dependent manner. [2] It is hypothesized that the autophagosomes may be a response step in the intracellular processing of Lipofectamine 2000/siRNA complexes. These inadvertent changes to the basal autophagic condition of the cells and subsequently, the overall cellular homeostasis may result in inaccurate observations and conclusions. This augments the need for an efficient, less toxic and cost-effective transfection reagent.
Studies have demonstrated that fusion peptides are more effective CPPs to deliver cargo. The peptide BP100-(KH)9 is the fusion of CPP BP100 and the polycationic peptide, (KH)9. Literature states that polycationic peptide is an efficient nucleic acid carrier, for example, the copolymer of lysine and histidine. It has the ability to promote buffering effect on the pre-lysosomal vesicle [3] besides playing an important role in the electrostatic interaction with negatively charged protein cargo [4] and in the ionic interaction with plasmid DNA (pDNA) [5, 6]. It has been successfully used to deliver protein and nucleic acids into plants [3]. The cargo, being negatively charged interacts with the polycationic peptide through ionic interactions, whereas the CPP interacts with fewer cargo molecules and is present on the surface of peptide-cargo complexes.
This fusion peptide has also been used to transform Gram-negative bacterial cells using large plasmid DNA (205 kb) with an efficiency equalling electroporation [7]. We propose to test the efficiency of this fusion peptide BP100(KH)9 in the transfection of shRNA cargo in animal cells thus establishing a wide range of applications.
Chimeric CPP BP100-(KH)9 delivering plasmid DNA into plant cell
CPP-mediated delivery of large sized plasmid DNA into bacterial cell
The CPP BP100(KH)9 has been successful in the delivery of proteins [4], plasmid [5] and double-stranded DNA [6] into cells. Thus we explored the efficiency of the transfection of the in-vitro synthesized RNA using this fusion peptide.
